Neonatal necrotizing enterocolitis (NEC) is a devastating inflammatory bowel disease affecting approximately 10% of premature infants born <1500gm. There is currently no means of predicting, preventing, or specifically treating this disease. The two primary risk factors for NEC are prematurity and bacterial colonization. This proposal is focused on understanding the function of the early intestinal microbiota. Specifically, the goal of this proposal is to elucidate the role of early human microbiota in the development of the immature intestine, and the mechanism by which this microbiota-host interaction alters susceptibility to NEC. Based on preliminary data demonstrating differences in the microbial communities of preterm infants who do or do not develop NEC, and differences in inflammatory signaling and barrier function associated with different preterm infant microbial communities, we hypothesize that optimal early microbial intestinal colonization enhances development of the immature preterm gut and exhibits stability despite clinical perturbations thus protecting against NEC. We propose to investigate the role of early microbial communities in infants who ultimately develop or do not develop NEC by using 1. Metagenomic sequencing to determine differences in functional microbial gene sequence of the overall community; 2. Germ-free mouse transfaunation to investigate the effect of the early microbial community on intestinal phenotype; 3. Chemostat bioreactors to measure the stability of the microbial community and volatile organic compounds (VOC) produced in response to clinically relevant perturbations; 4. Enteroid culture models to investigate mechanistic links between VOCs produced and inflammation or barrier function alterations in immature intestinal epithelial cells. Use of human preterm infant samples will ensure clinical relevance. We also propose to create defined experimental communities (DECs) of cultured organisms that are representative of the early microbiota from infants who do or do not go on to develop NEC. These DECs can form the basis for a potential future microbial ecotherapeutic intervention to optimize microbial colonization patterns in premature infants to promote normal intestinal development and prevent NEC in these vulnerable patients. These studies will also allow assessment of microbial community stability to enable understanding of how clinical interventions early in life are influencing this critical earl microbiota, to prompt informed clinical practice alterations.